Trehalose is a nonreducing disaccharide formed by an α,α-1,1-glucoside bond between two α-glucose units. It is widespread in organisms such as insects, crustaceans, fungi and bacteria, being used as their energy and carbon source. Trehalose, in contrast to reducing sugars, does not possess the toxicity caused by the Maillard reaction, which is a chemical reaction between amino acids in proteins and carbonyl group in reducing sugars and results in denaturation of the proteins (Elbein, A. D., et al. (2003) Glycobiology 13, 17R-27R). Moreover, trehalose has distinctive physiological properties not seen in any other sugars.
As an example, trehalose prevents osteoporosis by disturbing osteoclast differentiation (Nishizaki, Y., et al. (2000) Nutrition Research 20, 653). Also, it alleviates accumulation of abnormal proteins in nerve cells and muscle cells, caused by polyglutamine and polyalanine disease known as Huntington's disease (Tanaka, M., et al. (2004) Nat Med 10, 148-154.) and oculopharyngeal muscular dystrophy (Davies, J. E., et al. (2006) Hum Mol Genet. 15, 23-31), respectively. In addition, trehalose as a chemical chaperon or an antioxidant protects biomolecules such as proteins and the cellular membranes form stresses such as desiccation, heat, low temperature, and high and low oxygen (Crowe, J. H., et al. (2005) Integr Comp Biol 45, 810-820; Crowe, J. H., et al. (1987) Biochem J 242, 1-10; Crowe, J. H., et al. (1998) Annu Rev Physiol 60, 73-103; Elbein, A. D., et al. (2003) Glycohiology 13, 17R-27R; Benaroudj, N., et al. (2001) J Biol Chem 276, 24261-24267).
An advanced applied research on the bioactivity of trehalose is being conducted. For instance, by using trehalose as an anhydro-protectant, the Defense Advanced Research Projects Agency (DARPA) is developing a method to preserve the blood for transfusion in dry state (Brumfiel, G. (2004) Nature 428, 14-15). As a result, they have succeeded in preserving akaryotic platelet for almost two years by the technique that the platelets is heated to introduce trehalose into the cells through endocytosis, and then freeze-dried (Wolkers, W. F., et al. (2001) Cryobiology 42, 79-87).
However, platelet is so far the only successful example, since other karyotic cells have not yet been successfully desiccated. As for developing medication to cure Huntington's disease and osteoporosis for practical use (Couzin, J. (2004) Science 304, 816-817), not enough studies that apply the bioactivity of trehalose are being performed. These are attributed that trehalose is an impermeable molecule across the cellular membranes without heating. Hence, discovering the method to introduce trehalose into cells facilely without harming is the key to establish the uses of trehalose for basic and applied goals in karyotic cells and live bodies.
To date, several trials introducing trehalose into cells have been reported: introduction of bacterial trehalose biosynthetic enzyme genes (otsA and otsB) into cells increases intracellular trehalose (Guo, N., et al. (2000) Nat Biotechnol 18, 168-171); engineered switchable pores or extracellular nucleotide-gated channels were created in cellular membranes to allow trehalose uptake (Eroglu, A., et al. (2000) Nat Biotechnol 18, 163-167; Elliott, G. D., et al. (2006) Cryobiology 52, 114-127). Indeed, both techniques allow increasing intracellular concentration of trehalose. However, the former is hard to eliminate trehalose even when it is no longer necessary. This retention could cause ill effect because trehalose can prevent refolding of denatured proteins. The later enabled trehalose to move from the extracellular fluid into cells; however, undesired influx and efflux of other molecules probably occur simultaneously. In addition, introduction of trehalose into cells through spontaneous uptake such as pinocytosis has been attempted (Wolkers, W. F., et al. (2001) Cryobiology 42, 79-87); however, it is largely dependent on cell characteristics, so that uptake of a large amount of trehalose is not expected. Therefore, in order to easily and selectively introduce trehalose into cells, it is necessary to use trehalose-specific transporter localized in the cellular membranes.
Transporters promoting permeation of trehalose across cellular membrane have already been found from unicellular organisms such as bacteria, archaea and yeast. The transporters are active α-glucoside transporters such as MalEFGK2. (GeneBank no. P68187; TC: 3.A1.1) for bacteria and archaea (Boos, W. & Shuman, H. (1998) Microbiol Mol Biol Rev 62, 204-229), and MAL11/AGT1 (GeneBank no. P53048; TC: 2.A.1.1.11) for yeast (Stambuk, B. U., et al. (1996) Eur J Biochem 237, 876-881; Stambuk, B. U., et al. (1999) FEMS Microbiol Lett 170, 105-110; Han, E. K., et al. (1995) Mol Microbiol 17, 1093-1107). However, there are no reports that these transporters are utilized for introducing trehalose into cells in higher organisms. The reasons are thought to be as follows (Boos, W. & Shuman, H. (1998) Microbiol Mol Biol Rev 62, 204-229; Stambuk, B. U., et al. (1996) Eur J Biochem 237, 876-881; Stambuk, B. U., et al. (1999) FEMS Microbiol Lett 170, 105-110; Han, E. K., et al. (1995) Mol Microbiol 17, 1093-1107). (I) Intracellular energy might be consumed because the active transporters require either ATP hydrolysis or a favorable membrane potential as driving force to transport substrates. (II) Substrate selectivity of those transporters is relatively broad and includes alpha-glucosides such as trehalose, sucrose and maltose. (III) Coordinate expression of the four different genes needed for MalEFGK2 is difficult. (IV) The optimum pH for MAL11/AGT1 is acidic rather than neutral. (V) The direction of those transporters is only inward. Thus, these problems can be resolved by using the transporter that is facilitated and trehalose-specific transporter, is bidirectional between the external environment and cytosol, is governed by a single gene product, and is independent of pH and membrane potential.
The present invention was made in view of such a situation, and an objective of the present invention is to provide trehalose transporter gene and method of introducing trehalose into cells by using the gene. More specifically, the present invention provides a gene (Tret1) encoding a P. vanderplanki-derived facilitated trehalose transporter protein (TRET1), a vector carrying the gene, and a method for introducing trehalose into cells by using the gene.